Major histocompatibility (MHC) Class I antigens are polymorphic membrane glycoproteins that are necessary for antigen recognition by T-cells. To elucidate structure-function relationships of the antigen, we have employed site directed mutagenesis, in which DNA sequence of a Class I gene can be changed at desired positions. Expression and function of the mutant class I genes were examined in L cells after DNA mediated gene transfer. By using the mouse H-2Ld gene, we addressed the question of the role of highly conserved amino acids of the Class I antigen: those involved in the formation of disulfide bridges and in glycosylation. A mutant lacking the disulfide bridge in the domain most proximal to the membrane (3rd external domain) was generated by a single nucleotide substitution. We found that the mutant antigen is incapable of expressing the antigen in the plasma membrane, and that a large quantity of the antigen accumulates within the cytoplasm. This was vereified by immunocytochemistry and immunoprecipitation domain specific antibodies. These studies also indicated that the mutant antigen is glycosylated and associated with Beta-2 microglobulin. These observations led us to conclude that the disulfide bridge in this domain, conserved throughout Ig gene super family evolution, is essential for transport of the antigen from the endoplasmic reticulum to the plasma membrane. A mutant lacking all the N-linked glycosylation sites was produced by a 3 step mutagenesis. The resultant mutant antigen was found to be expressed on the cell surface, though at a reduced level, and is reactive both with cytotoxic T-cells and with a panel of antibodies specific for the wild type antigen. This data indicated that carbohydrate moieties are not essential for antigenicity and immune function of the antigens. Quantitative meaurement of cell surface versus intracellular mutant protein led us to conclude that carbohydrates are important primarily for efficient intracellular transport of the protein to the plasma membrane.